The present invention relates to methods and compositions for the direct detection of analytes using observable spectral changes in biopolymeric systems. In particular, the present invention allows for the direct colorimetric detection of analytes using color changes that occur in glycopolythiophene polymer systems in response to selective binding of analytes.
A major goal of analyte detection research is to develop inexpensive, fast, reliable, and sensitive detectors. Unfortunately, the technologies developed to date have only met some of these goals, and no single device has sufficiently attained a majority of them.
Classical detection methods such as liquid chromatography (LC), gas chromatography (GC), and supercritical fluid chromatography (SFC), in combination with mass spectrometry, are widely used and provide accurate identification of analytes and quantitative data. However, these techniques are time consuming, extremely expensive, require sample preconcentration, and are difficult or impossible to adapt to field use.
Biosensors (i.e., devices containing biological material linked to a transducing apparatus) have been developed to overcome some of the shortcomings of the classical analyte detection techniques. Many currently used biosensors are associated with transducer devices that use photometry, fluorimetry, and chemiluminescence; fiber optics and direct optical sensing (e.g., grating coupler); surface plasmon resonance; potentiometric and amperometric electrodes; field effect transistors; piezoelectric sensing; and surface acoustic wave (Krxc3xa4mer, J. AOAC Intern. 79: 1245 [1996]). However, there are major drawbacks to these devices, including their dependence on a transducing device, which prevents miniaturization and requires a power source. These disadvantages make such devices too complex, expensive, or unmanageable for many routine analyte detection applications such as field work or home use. Additionally, many of these devices are limited by the lack of stability and availability of the biological materials (e.g., proteins, antibodies, cells, and organelles).
The art remains in need of analyte detectors that provide the specificity of biosensors but also provide the cost-efficiency, stability, accuracy, reliability, reproducibility, and robustness that is lacking from available technologies. In particular, development of devices that can be miniaturized, that allow the detection of multiple analyte types, and that do not rely on an energy source would also be very beneficial, particularly for routine field work and home use.
The present invention relates to methods and compositions for the direct detection of analytes using observable spectral changes in biopolymeric systems. In particular, the present invention allows for the direct colorimetric detection of analytes using color changes that occur in glycopolythiophene polymer systems in response to selective binding of analytes.
The present invention provides biopolymeric materials comprising a plurality of polymerized monomers and one or more ligands, wherein the biopolymeric materials change color in the presence of analyte. In some embodiments, the ligands are selected from the group consisting of peptides, proteins, antibodies, receptors, channels, and combinations thereof, although the present invention contemplates all protein ligands (i.e., with protein being defined in its broadest sense). In other embodiments, the ligands are non-proteins (e.g., lectins, carbohydrates, glycolipids, phospholipids, and the like). However, the present invention is not limited to any particular ligand-analyte binding partners.
In particularly preferred embodiments, the biopolymeric materials comprise water-soluble glycopolythiophenes (e.g., containing sialic acid or mannose ligands) such materials have been synthesized by oxidative co-polymerization of methyl thiopheneacetate and thiophene-carbohydrate monomers.
In some embodiments, the inventive biopolymeric materials comprise three portions: a polymer, a spacer, and a ligand. Because of the adaptability of these assemblies, modifications may be made which give it great variety in application and design. The present invention is not limited to the following variations in some embodiments. Some preferred embodiments of the present invention employ variation in the polymer backbone, thereby producing different shapes of conjugation. This is accomplished through the addition of aromatic and or non-aromatic units (e.g., thiazole, pyrrole, selenophene phenyl unit, phenylene vinylene unit and diacetylene) as co-monomers without losing conjugation. Alternatively, the polymer backbone is altered by heterocyclic atoms used in place of carbon (e.g., N, O, and Se). Other embodiments of the present invention vary the length or composition of the spacer element. For example, almost any length spacer (e.g., one or more carbon atoms) having hydrophilic/lipophilic properties is permitted. The alteration of spacer length and composition is directed by observing the colorimetric responses obtained such that a desired response is reached. It is contemplated that the ability to vary spacer length and composition allows the polymer assemblies greater access to high molecular weight molecules (e.g., viruses, bacteria, and parasites). In preferred embodiments, neither the ligand, dopant, spacer, or polymer assembly comprises a lipid.
In still other embodiments, the ligand is varied according to the analyte to be detected. For example, in some embodiments, the ligand(s) employed include sugars, altered or naturally occurring polynucleotides (DNA, RNA, etc.), polypeptides, and other organic molecules capable of specifically binding to a receptor (e.g., cyclosporin, benzadiazapam, or serotonin uptake transporters, ACE), metal-complexes, and inorganic materials such as transition and lanthanide series metals.
It is not intended that the present invention be limited to one particular type of ligand molecule. A variety of ligand are contemplated. For example, the present invention provides for both protein and non-protein ligands. In some embodiments, protein ligands comprise antibodies or portions of antibodies, proteins, or polypeptides, and the like. In other embodiments that employ non-protein ligands, a number of non-protein molecules are contemplated (e.g., carbohydrates, nucleic acids, drugs, chromophores, antigens, chelating compounds, molecular recognition complexes, ionic groups, polymerizable groups, linker groups, electron donors, electron acceptor groups, hydrophobic groups, hydrophilic groups, receptor binding groups, polysacchrides (e.g., trisaccharides, tetrasaccharides, etc.) ganglioside GM1, ganglioside GT1b, sialic acid, and combinations thereof).
In alternative embodiments of the present invention, the portions of the ligand or monomer assemblies are manipulated to alter their shape and electronic conformation of the composition. For example, in some embodiments of the present invention, a carbohydrate ligand was placed next to the phenyl group of the glycopolythiophene assemblies to prevent neuraminidase cleavage of the O-linked glycosides of sialic acid and to provide tighter binding to Escherichia coli. Certain embodiments of the present invention were modified in this manner without losing binding properties.
Likewise, it is not intended that the present invention be limited to detecting any particular analyte. A variety of analytes are contemplated. In some embodiments, the analyte is selected from the group consisting of pathogens, drugs, receptor ligands, antigens, ions, hormones, blood components, disease indicators, cell components, antibodies, lectins, enzymes, organic solvents, volatile organic compounds, pollutants, and genetic material. Other embodiments are directed to pathogenic analytes, for example, viruses, bacteria, parasites, and fungi. In particular embodiments of the present invention, viral analytes are selected from the group consisting of influenza, rubella, varicella-zoster, hepatitis A, hepatitis B, herpes simplex, polio, small pox, human immunodeficiency virus, vaccinia, rabies, Epstein Barr, reoviruses, and rhinoviruses. However, it is understood that many other viruses or portions of viruses may be selected as suitable analytes. In still other embodiments, pathogenic analytes comprise bacterium, or portions of bacterium (e.g., E. coli, Mycobacterium tuberculosis, Salmonella, Chlamydia and Streptococcus). Alternative embodiments are directed to parasitic analytes (e.g., Plasmodium, Trypanosoma, Toxoplasma gondii, and Onchocerca) or portions of these parasites.
Still further embodiments of the present invention employ accessory molecules (i.e., dopants) that alter ligand-receptor binding. As used herein xe2x80x9calter ligand bindingxe2x80x9d encompasses both attenuating or enhancing ligand binding (the colorimetric response of the polymer assemblies upon the binding of an analyte may, or may not, be effected by the particular dopant selected). The present invention also contemplates a number of means, both covalent and noncovalent, for attaching these dopant molecules. For example, in some embodiments, these molecules are attached directly to the polymer assemblies, alternatively, these molecules are attached to the polymer assemblies via a linker. In still other embodiments, these molecules are attached to the ligand or receptor molecules themselves. In any event, the molecules are positioned relative to the polymer assemblies such that a desired effect is exerted on ligand binding or colorimetric response. Other embodiments of the present invention employ alternative physical schemes for altering ligand binding and colorimetric response (e.g., heat, pressure, irradiation, and the like).
In some embodiments of the present invention, the dopant material comprises surfactants, polysorbate, octoxynol, sodium dodecyl sulfate, polyethylene glycol, zwitterionic detergents, decylglucoside, deoxycholate, diacetylene derivatives, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylmethanol, cardiolipin, ceramide, cholesterol, steroids, cerebroside, lysophosphatidylcholine, D-erythroshingosine, sphingomyelin, dodecyl phosphocholine, and N-biotinyl phosphatidylethanolamine.
In particularly preferred embodiments of the present invention, the biopolymeric materials are designed to produce freely soluble polymers in aqueous solution and to increase inter-chain interactions so that the compositions (e.g., glycopolythiophenes, polythiophenes, and thiophenes) are more sensitive to external stimuli. This unique feature distinguishes this invention from other polymers. However, the present invention is not intended to be limited to any particular configuration. Various configurations are envisioned. In one embodiment, the polymer assemblies comprise linear glycopolymers. In other embodiments, the polymer assemblies are configured as films, or vesicles. Still further embodiments contemplate compositions and methods employing immubolized polymers (e.g., various substrates, including microtitter plates, tubes, and wells).
The present invention is not intended to be limited to any particular method of polymer assembly or synthesis. Indeed, a number of polymer assembly synthesis techniques are contemplated. For example, in some embodiments, the polymer assemblies are synthesized by oxidative polymerization by the described FeCl3 method. In alternative embodiments, the polymer assemblies are synthesized via graft conjugation of carbohydrates by the described peptide coupling method.
It is not intended that the present invention be limited to glycopolythiphene monomers. A variety of polymerizable monomers are contemplated. In one embodiment, thiophene monomers are used. In another embodiment, polythiophene monomers are used.
In some particularly preferred embodiments of the present invention, the compositions further comprise one or more spacer molecules. Suitable spacer molecules can be hydrophilic or hydrophobic. In some of these embodiments, the spacer molecule comprise from 1-1,000 carbon atoms, preferably from 100-500 carbon atoms, more preferably from 20-50 carbon atoms, and most preferably from 5-10 carbon atoms. Additionally, in some embodiments, one or more covalent bonds attach one or more ligands to the biopolymeric materials (e.g., amine bonds, sulfide bonds, thiol bonds, aldehyde bonds, glycosidic bonds, and peptide bonds). In some of these embodiments, also comprise one or more spacer molecules.
In preferred embodiments of the present invention, a colorimetric response is obtained from conformational changes in the biopolymeric assemblies. While an understanding of the mechanism is not important to practice of the present invention, the colorimetric response is believed to result from stress induced in the polymer through the binding of an attached ligand and its analyte.
The present invention also provides methods of detecting the presence of an analyte in a sample, comprising: a) providing: i) biopolymeric materials comprising a plurality of monomers and one or more ligands wherein said biopolymeric materials change color in the presence of an analyte; and ii) a sample suspected of containing an analyte; a) contacting said biopolymeric materials with said sample; and b) detecting a color change in said biopolymeric materials. Indeed, the present invention contemplates various methods and kit embodiments for detecting the presence of analytes using the novel colorimetric biopolymeric materials disclosed herein. For example, the compositions and methods of the presention are readily suited for assays employed to discover various reaction inhibitors. Moreover, the compositions and methods of the present invention are fully scale-able for uses requiring high throughput screening techniques such as drug development, analytical chemistry, genomics, and proteomics.
The present inventive assemblies can also be applied to the manufacture of environmental biosensors for the detection of air and water contaminants and contaminants in food and beverages. In still further embodiments, the inventive compositions are incorporated with surgical instruments or medical consumables (e.g, bandages and wound dressings, catheters, etc.) such that clinicians and other helathcare workers can moniter the presence of an analyte (e.g., a bacterial toxin, or metabolite). In still further embodiments, the compositions and methods of the present invention are devised for the home health care nmarkets (e.g., pregnancy tests, glucose and insulin level monitoring).